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. 2020 Jan 14:12:265-275.
doi: 10.2147/CMAR.S226410. eCollection 2020.

ERα36 as a Potential Therapeutic Target for Tamoxifen-Resistant Breast Cancer Cell Line Through EGFR/ERK Signaling Pathway

Guangliang Li  1 , Jing Zhang  2 , Zhenzhen Xu  2 , Zhongqi Li  2
Affiliations

ERα36 as a Potential Therapeutic Target for Tamoxifen-Resistant Breast Cancer Cell Line Through EGFR/ERK Signaling Pathway

Guangliang Li et al. Cancer Manag Res. .

Abstract

Background: Acquired tamoxifen resistance is one of the major barriers to the successful treatment of breast cancer. Recently, overexpression of ERα36 was demonstrated to be a potential mechanism for the generation of acquired tamoxifen resistance. This study aims to evaluate the possibility of ERα36 being a therapeutic target for tamoxifen-resistant breast cancer.

Methods: A tamoxifen-resistant cell subline (MCF-7/TAM) was established by culturing MCF-7 cells in medium plus 1 μM tamoxifen over 6 months. Colony-forming assay was used to determine the sensitivity of MCF-7/TAM cells to tamoxifen. Stable transfection was used to knockdown ERα36 expression in MCF-7/TAM cells. MTT assay and Transwell migration assay were used to assess the in vitro proliferation and migration, respectively. Nude mouse tumorigenicity assay was used to evaluate in vivo tumorigenicity. Western blot analysis and quantitative real-time PCR (qRT-PCR) were used to examine the expression of ERα36, ERα, EGFR and phosphorylated ERK1/2. The dual-luciferase reporter assay was used to determine the effect of ERα36 on the activity of EGFR-promotor.

Results: MCF-7/TAM cells possessed greatly increased ERα36 expression and EGFR expression and exhibited significantly increased in vitro proliferation and migration ability, as well as increased in vivo tumor growth ability, compared to parental MCF-7 cells. Knockdown of ERα36 expression inhibited in vitro proliferation and migration, as well as in vivo tumor growth ability of MCF-7/TAM cells. ERα36 regulated EGFR expression at the transcriptional level, and knockdown of ERα36 in MCF-7/TAM cells downregulated EGFR expression and then blocked EGFR/ERK signaling pathway.

Conclusion: Knockdown of ERα36 inhibits the growth of MCF-7/TAM cells in vitro and in vivo by blocking EGFR/ERK signaling pathway. ERα36 may be a candidate therapeutic target for the treatment of tamoxifen-resistant breast cancer.

Keywords: EGFR/ERK signaling; ERα36; acquired tamoxifen resistance; breast cancer.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Schematic overview of the experimental design and workflow of experiment in this study.
Figure 2
Figure 2
Tamoxifen-resistant MCF-7 cells (MCF-7/TAM) exhibit tamoxifen-insensitive growth and increased expression of ERα36. (A). Cells were seeded at 500 cells per well in 6-well plates, incubated in medium containing DMSO (vehicle) or 1 μM tamoxifen (TAM) and counted after 2 weeks. (B). Column: means of three independent experiments; bars, SEM. **Represents P<0.01 (the multiple two tailed Student's t-tests p-value in the DMSO group is 0.000008 and in the TAM group is 0.000001). (C). Cell viability rate of tamoxifen-treated MCF-7 cells and MCF-7/TAM cells was calculated. Data presented are means ± SEM of three independent experiments. ** represents P<0.01 (the two tailed Student's t-test p-value is 0.002). (D). Western blot analysis of the protein levels of ERα36 and ERα in MCF-7 cells and MCF-7/TAM cells. β-actin was used as the loading control. All experiments were repeated at least 3 times, and the representative results are shown.
Figure 3
Figure 3
MCF-7/TAM cells exhibit significantly increased proliferation and migration in vitro. (A). Relative cell proliferation rate of parental MCF-7 cells and MCF-7/TAM cells in routine RPMI 1640 medium with phenol red indicator supplemented with 10% FBS that contains estrogen was determined via MTT assay. Data presented are means ± SEM of three independent experiments. **Represents P<0.01 (the multiple two tailed Student's t-tests p-value is 0.00005 at day 3 and 0.00004 at day 4). (B). The in vitro migratory ability of MCF-7 and MCF-7/TAM cells in routine RPMI 1640 medium with phenol red indicator supplemented with 10% FBS that containing estrogen was determined by Transwell migration assay. Representative photomicrographs of migration assay results were taken under 200 original magnifications. (C). Cells migrated through the membrane were viewed at ×200 magnifications, counted in 5 independent visual fields per transwell membrane. Cell numbers were presented as values of means ± SEM of triplicate experiments. **Represents P<0.01 (the t test p-value is <0.000001).
Figure 4
Figure 4
Knockdown of ERα36 expression inhibits in vitro proliferation and migration of MCF-7/TAM cells. (A) Whole cellular protein extracts of MCF-7/TAM cells transfected with pcDNA3.1(+) vector or pcDNA3.1(+)-6mi36 were subjected to Western blot analysis using an anti-ER-α36 antibody. β-actin was used as the loading control. All experiments were repeated at least 3 times, and the representative results are shown. (B). Relative cell proliferation rate of MCF-7/TAM-V cells and MCF-7/TAM-mi36 cells in routine RPMI 1640 medium with phenol red indicator supplemented with 10% FBS that contains estrogen was determined using MTT assay. Data presented are means ± SEM of three independent experiments. **Represents P<0.01 (the multiple t tests p-value is 0.000013 at day 3 and <0.000064 at day 4). (C). The in vitro migratory ability of MCF-7/TAM-V and MCF-7/TAM-mi36 cells in routine RPMI 1640 medium with phenol red indicator supplemented with 10% FBS that containing estrogen was determined by Transwell migration assay. Representative photomicrographs of migration assay results were taken under 200 original magnifications. (D). Cells migrated through the membrane were viewed at ×200 magnifications, counted in 5 independent visual fields per transwell membrane. Cell numbers were presented as values of means ± SEM of triplicate experiments. **Represents P<0.01 (the two tailed Student's t-test p-value is <0.000001).
Figure 5
Figure 5
Knockdown of ERα36 expression inhibits in vivo tumor growth of MCF-7/TAM cells in nude mice. (A). Parental MCF-7 cells and MCF-7/TAM cells were implanted into the left mammary fat pad of the female mice without exogenous estrogen. Photographs of tumors were taken. (B). The tumorigenicity was examined by measurement of tumor size. The data represent the means ± SEM observed in 5 mice in each group. (C). MCF-7/TAM-V cells and MCF-7/TAM-mi36 cells were implanted into the left mammary fat pad of the female mice without exogenous estrogen. Photographs of tumors were taken. (D). The tumorigenicity was examined by measurement of tumor size. The data represent the means ± SEM observed in 5 mice in each group.
Figure 6
Figure 6
Knockdown of ERα36 expression deactivates EGFR/ERK signaling pathway in MCF-7/TAM cells. (A). Western blot analysis of the protein levels of EGFR, phosphorylated ERK1/2 and total ERK1/2 in MCF-7 cells and MCF-7/TAM cells. β-actin was used as the loading control. All experiments were repeated at least 3 times, and the representative results are shown. (B). Western blot analysis of the protein levels of EGFR, phosphorylated ERK1/2 and total ERK1/2 in MCF-7/TAM-V cells and MCF-7/TAM-mi36 cells. β-actin was used as the loading control. All experiments were repeated at least 3 times, and the representative results are shown.
Figure 7
Figure 7
ER-α36 regulates EGFR expression at the transcriptional level. (A). Relative mRNA level of EGFR in MCF-7/TAM cells was determined by real-time qPCR. β-actin gene was used as an endogenous control for normalization. Results showed are means ± SEM of three independent reactions. ** represents P<0.01 (the t test p-value is 0.00006). (B). Relative mRNA level of EGFR in MCF-7/TAM-mi36 cells was determined by real-time qPCR. β-actin gene was used as an endogenous control for normalization. Results showed are means ± SEM of three independent reactions. **Represents P<0.01 (the t test p-value is 0.0014). (C). Relative mRNA level of EGFR in MCF-7/ERα36 cells was determined by real-time qPCR. β-actin gene was used as an endogenous control for normalization. Results showed are means ± SEM of three independent reactions. **Represents P<0.01 (the t test p-value is 0.001). (D). Whole cellular protein extracts of MCF-7 cells transfected with pcDNA3.1(+) vector or pcDNA3.1(+)-ERα36 were subjected to Western blot analysis using an anti-ERα36 antibody. β-actin was used as the loading control. All experiments were repeated at least 3 times, and the representative results are shown. (E). Relative light unit caused by pcDNA3.1(+)-NC + pGL3-promotor + pRL/TK vector (MCF-7/V+pGL3-promotor+pRL/TK, Column A), pcDNA3.1(+)-ERα36 + pGL3-promotor+pRL/TK vector (MCF-7/ERα36+pGL3-promotor+pRL/TK, Column B), pcDNA3.1(+)-NC + pGL3-EGFR-promotor + pRL/TK vector (MCF-7/V+pGL3-EGFR-promotor+pRL/TK, Column C), pcDNA3.1(+)-ERα36 + pGL3-EGFR-promotor + pRL/TK vector (MCF-7/ERα36+pGL3-EGFR-promotor+pRL/TK, Column D) by luciferase assay. ** represents P<0.01 (the multiple two tailed Student's t-tests p-value is 0.717085 in Column A vs Column B; 0.000001 in Column A vs Column C; 0.000003 in Column A vs Column D; 0.000003 in Column B vs Column D; 0.000042 in Column C vs Column D). (F). Cellular protein extracts of MCF-7 cells transfected with pcDNA3.1(+)-NC (MCF-7/V) or pcDNA3.1(+)-ERα36 (MCF-7/ERα36) were subjected to Western blot analysis using an anti-ERα36 antibody. β-actin was used as the loading control.
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